1. Technical Field
The present invention is generally related to detection of nucleic acid sequences, as well as methods for rapid identification of target sequences in mixed nucleic acid samples.
2. Description of the Related Art
Rapid and accurate detection of nucleic acid sequence has become increasingly important. For example, threats of biological warfare and terrorism impose a need for rapid identification of specific pathogens in samples found on the battlefield, at border crossings, and in the work environment generally. In addition, infectious disease accounts for approximately 7% of human mortality in developed nations, and as much as 40% in the developing world. Rapid and accurate identification of the causative pathogen could result in increased survival of infected patients and enable better containment of outbreaks. Unfortunately, traditional microbiology techniques (e.g., cell culturing) for identifying biological agents can take days or even weeks, often delaying a proper course of action. Antibody based assays can be done quickly and easily, but often lack sensitivity and/or specificity.
A desirable alternative to traditional approaches would have one or more of the following characteristics: high specificity (>99.9%) and sensitivity (<1000 copies); high-level multiplexing (>100 targets); automated sample preparation; fully integrated processes on disposable reagent cartridge; sample-to-result in less than 15 minutes; rapid reconfiguration for addition of new biomarkers; flexible targeting; and/or have an associated compact, low-cost field deployable instrument. Unfortunately, there are currently no technologies on the market that satisfy these criteria.
Probe hybridization arrays can measure a broad spectrum of nucleic acid targets (I. Biran, D. R. Walt, and J. R. Epstein, “Fluorescence-based nucleic acid detection and microarrays,” Analytica Chimica Acta, vol. 469, no. 1, pp. 3-36.1), but typically have limited sensitivity of 105 to 106 target molecules and are also limited by diffusion and nonspecific binding. Bead-based methods of improving sensitivity to attomolar levels have been devised (J. Nam, S. I. Stoeva, and C. A. Mirkin, “Bio-Bar-Code-Based DNA Detection with PCR-like Sensitivity,” Journal of the American Chemical Society, vol. 126, no. 19, pp. 5932-5933, May. 2004; S. I. Stoeva, J. Lee, J. E. Smith, S. T. Rosen, and C. A. Mirkin, “Multiplexed Detection of Protein Cancer Markers with Biobarcoded Nanoparticle Probes,” Journal of the American Chemical Society, vol. 128, no. 26, pp. 8378-8379, July 2006). Bead-based methods to reduce nonspecific signal have also been devised (S. P. Mulvaney et al., “Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics,” Biosensors & Bioelectronics, vol. 23, no. 2, pp. 191-200, September 2007).
Specificity and sensitivity limitations of probe hybridization methods have been improved by using two probes that hybridize and ligate to produce a single product with unique electrophoretic drag (U.S. Pat. No. 4,883,750). This approach has provided two advantages: (i) the single product helps isolate its signal from other background, and (ii) the specificity of the ligase ensures high fidelity within five bases of the ligation site. Further improvements have involved adding fluorescent identifiers to probes (E. S. Winn-Deen and D. M. Iovannisci, “Sensitive fluorescence method for detecting DNA-ligation amplification products,” Clinical Chemistry, vol. 37, no. 9, pp. 1522-1523, September 1991; U.S. Pat. No. 5,514,543). Higher information identifiers have been proposed that require amplification in order to obtain sufficient signal to identify the target (PCT Publication WO 2010/115100; U.S. Pat. No. 7,320,865).
DNA sequencing can also be used for the identification of pathogens. Because each pathogen's DNA sequence is unique, sequencing allows for the identification of any number of different pathogens. Currently, most diagnostic DNA sequencing is performed using the chain termination method developed by Frederick Sanger. This technique, termed “Sanger Sequencing,” uses sequence specific termination of DNA synthesis and fluorescently modified nucleotide reporter substrates to derive sequence information. However, this method comprises a modified polymerase chain reaction (PCR) which makes this approach time consuming and expensive. Furthermore, PCR cannot simultaneously amplify many pathogen probe targets in a single reaction which requires samples to be split into many parallel reaction paths, increasing complexity and cost while reducing sensitivity. As a result, new sequencing methods, such as products from Illumina (San Diego, Calif.) and Life technologies (Carlsbad, Calif.), are displacing traditional methods. These technologies, however, are still largely reliant on PCR and use expensive and complex equipment that are not appropriate for rapid, low cost on-site detection.
Accordingly, while significant advances have been made in the field of nucleic acid detection generally, there is still a need in this field for techniques and corresponding devices that enhance or otherwise improve on the current state of the art, particularly with regard to sensitivity, specificity, high multiplexing, rapid result time, portability and/or cost. The present invention fulfills some or all of these needs, and provides further related advantages as evident upon review of the attached drawings and following description.